摘要
目的探讨激活miR-101-3p/EZH2通路在增敏索拉非尼抗肝癌HepG2细胞中的意义。方法将人肝癌细胞HepG2分成对照组(DMSO)和索拉非尼处理组(10μmol/L),处理24 h后,定量PCR检测miR-101-3p表达水平变化;生物信息学预测miR-101-3p结合的靶基因;在肝癌细胞中过表达miR-101-3p后Western blot和定量PCR检测zeste同源蛋白2增强子(enhancer of zeste homolog 2 protein,EZH2)表达水平变化;双荧光素酶报告基因实验验证miR-101-3p和EZH2的靶向关系;Western blot检测索拉非尼处理对EZH2表达水平的影响;在HepG2细胞中过表达miR-101-3p或添加EZH2抑制剂EPZ-6438后联合索拉非尼处理,CCK-8检测对HepG2细胞存活的影响。结果索拉非尼处理后HepG2细胞中miR-101-3p表达水平显著降低(P<0.05);miR-101-3p可在HepG2细胞中下调EZH2的表达(P<0.05);miR-101-3p与EZH2 mRNA 3'-UTR存在直接结合(P<0.05);索拉非尼处理后HepG2细胞中EZH2表达水平显著升高(P<0.05);过表达miR-101-3p或EZH2抑制剂EPZ-6438均可增加索拉非尼对HepG2细胞杀伤的敏感性(P<0.05)。结论激活miR-101-3p/EZH2通路可增敏索拉非尼的抗肝癌细胞HepG2效果。
Objective To investigate the effect of activating the miR-101-3 p/EZH2 pathway on sorafenib sensitivity of HepG2 cells.Methods HepG2 cells treated with sorafenib(10μmol/L)for 24 h were examined for protein expression of enhancer of zeste homolog 2(EZH2)using Western blotting and for mRNA expression of EZH2 and miR-101-3 p using RT-qPCR.The interaction between miR-101-3 p and EZH2 mRNA was analyzed through informatics analysis and verified using dual luciferase reporter assay.HepG2 cells were transfected with miR-101-3 p mimic or treated with the EZH2 inhibitor EPZ-6438 followed by treatment with sorafenib for 24 h,and the change in cell proliferation was assessed using CCK-8 assay.Results Sorafenib obviously decreased the expression of miR-101-3 p in HepG2 cells(P<0.05).Informatics analysis showed that miR-101-3 p could bind to the 3’-UTR of EZH2 mRNA,and transfection with miR-101-3 p significantly inhibited the expression of EZH2 in HepG2 cells(P<0.05).Sorafenib treatment significantly increased the expression of EZH2 in HepG2 cells(P<0.05).Both miR-101-3 p over-expression and EPZ-6438 treatment resulted in significantly increased sorafenib sensitivity of HepG2 cells(P<0.05).Conclusion Activation of the miR-101-3 p/EZH2 pathway can enhance sorafenib sensitivity of HepG2 cells.
作者
张越婷
肖翰希
孙梁博
李涛
陈岺曦
闫小晶
何凤田
连继勤
ZHANG Yueting;XIAO Hanxi;SUN Liangbo;LI Tao;CHEN Lingxi;YAN Xiaojing;HE Fengtian;LI AN Jiqin(Department of Biochemistry and Molecular Biology,College of Basic Medical Sciences,Army Medical University(Third Military Medical University),Chongqing,400038,China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2020年第22期2195-2201,共7页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(81572375)
重庆市自然科学基金面上项目(CSTC2018jcyjA2018)。
关键词
MICRORNA
索拉非尼
耐药
肝癌
EZH2
microRNA
sorafenib
drug resistance
hepatoma
enhancer of zeste homolog 2